1. Field of the Invention
The present invention is in the field of methods for determination of cardiovascular risk factors and protective factors in biological samples.
2. Description of Related Art
Cardiovascular risk factors include L-arginine (ARG), methylated arginines and lysines, isoprostanes, derivatives and metabolites of isoprostanes, enzymes like matrixmetalloproteinases (MMPs), or vitamins. However, this listing is understood to be exemplary and by no means complete.
Among the cardiovascular risk factors, the methylated arginines and the endogenous inhibitors asymmetric dimethylarginine (ADMA) and monomethyl arginine (MMA) are especially important. Their importance is based on the fact that they are essential for the regulation of nitric oxide (NO) synthesis in the human body. NO in turn is essential in several physiological settings, i.e. homeostasis of the cardiovascular system. Imbalance of NO supply and requirement is regarded as the initial step for pathophysiological changes that eventually lead to cardiovascular diseases like atherosclerosis, hypertension, and thromboembolic disorders. Accumulating evidence suggests that such NO imbalance of homeostasis are mainly linked to ADMA. ADMA and MMA originate from protein arginine methylation, they are released from proteins during protein degradation. Thus far, circulating ADMA was shown to be altered in patients suffering from cardiovascular diseases. Elevated plasma ADMA concentrations are found in various clinical settings ranging from renal failure to atherosclerosis, hypertension diabetes, preeclampsia, alzheimer's disease and even depression or schizophrenia. Moreover, in patients with cardiovascular disease elevated plasma ADMA concentrations independently predict progression of atherosclerosis and mortality.
All currently applied analytical techniques for the determination of cardiovascular risk factors rely upon detection of these factors in plasma, serum and urine specimens. In human blood and urine specimens, the parameters ARG, ADMA, SDMA and MMA together with the biochemical parameters such as C-reactive protein (CRP), isoprostanes, MMPs, Myeloperoxidase, HDL, LDL or total cholesterol have been evaluated to assess cardiovascular risk (Sydow: Z Kardiol 2001, Böger: Cardiovasc Res 2003, Ridker: Am J Cardiol 2003, Schwedhelm: Clin Chem Lab Med 2003). However, using human blood requires rapid separation of cellular blood constituents to obtain serum or plasma and thereby avoiding sample degradation. Thus, sample preparation steps are required which necessitate further equipment at the site of sample collection, such like centrifuges, pipettes, refrigerators, etc. The need of sample preparation makes the sampling elaborate and time-consuming.
The most efficient and precise methods utilized today for quantifying ARG and its methylated analogs are based on LC-MS or LC-MS/MS, although various other methods for determination of these important cardiovascular risk factors have been developed: spectrophotometry, capillary electrophoresis, liquid chromatography, GC-MS or immunoassays like ELISA. Equipment for LC-MS or LC-MS/MS is only available in few large laboratories. Thus, human blood samples commonly have to be shipped from physicians offices or pharmacies to the laboratory which results in samples to be sent in frozen state and which renders the step of sample preparation crucial for the quality of the analysis.
Due to the importance of ARG and its methylated analogs as cardiovascular risk factors, there is a need for a robust method of sample preparation which avoids degradation of analytes prior to quantitation in the clinical chemical laboratory and which allows a large number of samples to be routinely measured.